Individuals suffering from diabetes mellitus have an abnormally high blood sugar level generally because the pancreas does not secrete sufficient amounts of the active hormone insulin into the bloodstream to regulate carbohydrate metabolism. If an abnormally high blood sugar level, known as a hyperglycemic condition, is allowed to continue for prolonged periods, the individual will suffer from the chronic complications of diabetes, including retinopathy, nephropathy, neuropathy and cardiovascular disease. Studies indicate that diabetic patients who are able to maintain near normal glycemic control greatly reduce the likelihood of these dire complications. Therefore, several tests have been developed to measure and control glycemic condition.
One common medical test to control glycemic condition is the direct measurement of blood glucose levels by diabetics. Because blood glucose levels fluctuate significantly throughout a given day, being influenced by diet, activity, and treatment, depending on the nature and severity of the individual case, some patients measure their blood glucose levels up to seven times a day. Based on the observed pattern in the measured glucose levels, the patient and physician together make adjustments in diet, exercise and insulin intake to better manage the disease. Clearly, this information should be available to the patient immediately.
However, because of the frequent fluctuation of glucose levels in a given day, tests which are independent of a patient's diet, activity, and/or treatment and which provide longer term indications of blood glucose levels have also been developed. These tests measure the concentration of glycated proteins or “protein-bound glucose” (PBG). Proteins, such as those present in whole blood, serum and other biological fluids react with glucose, under non-enzymatic conditions, to produce glycated proteins. The extent of the reaction is directly dependent upon the glucose concentration of the blood.
One of the first glycated protein tests developed measures glycated hemoglobin, namely Hemoglobin A1c (HbA1c), which reflects glycemic control over approximately a 2 to 3 month period. Other such tests measure serum proteins, such as total glycated serum protein, or a specific glycated serum protein, namely glycated albumin. Glycated albumin reflects an intermediate glycemic control over approximately a 2 to 3 week period.
Yet another way to indirectly assess blood sugar concentration is to analyze glycated protein concentration. The plasma proteins are glycated in vivo by a non-enzymatic reaction between glucose and available amino groups of blood proteins, principally the γ-amino groups of lysine residues and the α-amino groups of the protein's terminal amino acid. The glucose binds to an amino group of the protein to form a Schiff base, i.e., aldimine, that undergoes molecular rearrangement to form a stable ketoamine. In the art, such ketoamines are generically known as “fructosamines.” The degree of protein glycation and fructosamine formation is directly proportional to blood glucose concentration. Measurement of serum or plasma glycated protein levels is useful for monitoring diabetic control because glycated protein concentrations in serum or plasma reflect an average of blood glucose level over approximately a half month period.
One currently employed assay that provides accurate determinations of blood glycated proteins, levels is the GlyPro™ assay currently marketed by Genzyme Corporation, where this assay is described in U.S. Pat. No. 6,008,006. While this assay provides accurate results, it is performed in a clinical lab by a trained technician with a sophisticated instrument, and is therefore not suitable for home or physician office use.
U.S. Pat. Nos. 5,470,752; 5,695,949 and 5,725,774 describe a multilayer reagent test strip for fructosamine quantification, where the test strip is designed for home or physician office use. However, measurements provided by the test strips described herein tend to be inaccurate, as substances in the fluid sample other than the fructosamine analyte also react with the signal producing system and affect the signal generated thereby, leading to inaccuracies in the ultimate fructosamine quantification achieved with such test strips.
Accordingly, there is continued interest in the development of additional multilayer reagent strips formats that are suitable for glycated protein quantification, where the test strips are suitable for use in the home or physician office and provide for the highly accurate measurements achieved with the currently employed clinical laboratory based protocols.
Relevant Literature
United States Patents of interest include: U.S. Pat. Nos. 5,470,752; 5,695,949; 5,725,774; 6,008,006. Also of interest are: WO 96/31270; WO 96/31619; WO 96/34977; EP 821064; and EP 737744.